The F-plasmid protein CcdB kills E. coli cells that express it, unless they also express its inhibitor CcdA. CcdB acts by inhibiting DNA gyrase and causing it to cleave chromosomal DNA. We have studied the interaction of CcdB with DNA gyrase. We have confirmed that CcdB can induce DNA cleavage by gyrase and show that this cleavage reaction requires ATP hydrolysis when the substrate is linear DNA, but is independent of hydrolysis when negatively-supercoiled DNA is used. The 64 kDa domain of the gyrase A protein, which can catalyse DNA cleavage in the presence of the B protein and quinolone drugs, is unable to cleave DNA in the presence of CcdB unless the C-terminal 33 kDa domain of the gyrase A protein is also present. CcdB-induced DNA cleavage by gyrase requires a minimum length of DNA (>160 bp) whereas in the presence of quinolone drugs gyrase can cleave much shorter DNA molecules. CcdB, like quinolones, forms a complex with gyrase which can block transcription by RNA polymerase. A model for the interaction of CcdB with gyrase involving the trapping of a post-strand-passage intermediate is suggested. Thus, CcdB can stabilise a cleavage complex between DNA gyrase and DNA in a manner distinct from quinolones but, like the quinolone-induced cleavage complex, the CcdB-stabilised complex can also form a barrier to the passage of polymerases.